Copolymer-grafted polyolefin substrate having antimicrobial properties and method for grafting

ABSTRACT

A method for grafting a copolymer onto a polyolefin substrate includes the following steps:
     (a) irradiating the substrate with ionizing radiation to obtain an activated polyolefin substrate,   (b) bringing into contact the activated polyolefin substrate with a mixture of at least two compounds in distilled water including:
       (i) from 10 to 40% by volume, related to the total volume of the reaction medium, of a hydrophilic unsaturated monomer selected from monomers having the formula:   
       

     
       
         
         
             
             
         
       
     
     wherein R 1  is H or methyl, R 2  is —COOH, —NH 2 , —CON(R 3 ) 2 , and R 3  is H or methyl,
         (ii) from 20 to 50% by volume, related to the total volume of the reaction medium, of an antimicrobial agent having an average molecular weight of at least 200 g·mol −1 , to thereby form a copolymer-grafted polyolefin substrate. Copolymer-grafted polyolefin substrates obtained by this method and a packaging material including copolymer-grafted polyolefin substrate are disclosed.

The present invention relates generally to grafted polyolefin substrateshaving antimicrobial properties and more particularly to a method forgrafting a copolymer having antimicrobial properties onto polyolefinsubstrates by radiation induced graft polymerization.

Polymeric materials have acquired an enormous potential in the packagingindustry where products have to be preserved for a long duration withoutaltering their inherent physical characteristics. One such requirementis the development of a proper packaging so that the packed productremains protected against any microbial infection notably during thestorage span. Consequently, control of microbial infection is a majorconcern for many applications involving, for instance, the preparationand the storage of food, medical and biopharmaceutical products.

This is why a substantial amount of work is being directed to theincorporation of antimicrobial agents into or onto the surface of suchpolymeric materials. Thus polymeric materials acquire ability to kill,inhibit or delay the growth and metabolism of micro-organisms that maybe present in the packed product or packaging material.

Some compounds inherently have antimicrobial properties. For example,quaternary ammonium salts are known for their bactericide and/orbacteriostatic activities and polyethylene glycol compounds are knownfor their repellent activity. These kinds of compounds have been graftedon various substrates.

Another approach involves modifying the surface properties of thesubstrate to decrease or to suppress adhesion of micro-organisms(anti-adhesives properties). In the case of certain polymers, notablypolyolefins, surfaces are so constituted that they adsorbmicro-organisms spontaneously. Such adsorption from aqueous solution ispromoted primarily by two types of physical forces, electrostaticattraction and hydrophobic interaction. Polyolefin surfaces arehydrophobic. Micro-organisms usually have hydrophobic domains.Therefore, the micro-organism is attracted to the polyolefin surface atleast by hydrophobic interactions between hydrophobic domains of themicro-organism and the polyolefin surface. This is described in, forexample, Surface and Interfacial Aspects of Biomedical Polymers, Ed. J.D. Andrade, Plenum Press (1985), Vol. 2, p. 81. In order to suppressadhesion of micro-organisms, efforts have been made to find coatingswhich resist to the adsorption of micro-organisms. These coatings have amicroscopic surface that lacks the structural characteristicsresponsible for adhesion.

There are several methods for conferring these antimicrobial propertiesonto polymer surface as well as into the bulk. These methods generallyinvolve modifying the polymer composition by chemically altering itsmacromolecular structure either by immobilizing an antimicrobial agentonto the polymer surface or blending the polymer with an antimicrobialagent.

Among the known techniques some of them lead to changes into the bulk ofthe polymer. These changes have a major drawback because the polymer mayundergo considerable physical structure changes notably in terms ofcrystallinity, mechanical strength and thermal stability.

A number of surface modification techniques have also been used todevelop surfaces with antimicrobial properties. Some of them are silvercoating, surface immobilized polyethylene oxide, surface thiocyanation,and surface modification by various gas plasma (such as oxygen andargon) and plasma immersion ion implantation.

Radiation induced graft polymerization has also been proved efficient tointroduce different functionalities onto polymer surface (B. Gupta, N.Anjum, R. Jain, N. Revagade, H. Singh, J. Macromol. Sci. 44 (2004) 275).This surface modification technique involves forming active sites on apolymeric substrate by irradiation with high-energy ionizing radiationeither in the presence of a monomer capable of grafting to the activesites, or followed by a contacting step with a monomer. The freeradicals produced in the polymer, as a result of the irradiation, act asinitiators for the polymerization of the monomer, as well as activesites for grafting.

For example, vinyl monomers have been polymerized onto various inertpolymers like PE, PP, PET, LLDPE and HDPE in different shape of polymers(sheet, film, fibre, hollow fibre, fabrics etc.) by radiation inducedgraft polymerization.

More particularly, surface modification of polypropylene sheets carriedout by radiation induced polymerisation of pure and diluted acrylic acidhas been disclosed in the document Anjum, N. and al. “Surface designingof polypropylene by critical monitoring of the grafting conditions” JAppl Polym Sci, 2006. According to this document the grafting isstrongly dependent on the monomer dilution in the reaction medium.Notably, in the presence of pure monomer, the grafted chains remainconfined to the surface. However, in the case of diluted monomer (80%monomer), water acts as the vehicle for the monomer diffusion within thesurface layers. Therefore, most of the grafts tend to move inside andonly little grafts remain on the surface. Furthermore, in the case ofsurface modified by pure monomer, the degree of grafting has to be veryhigh in order to get hydrophilic surfaces. But high degree of graftingaffects the physical structure of polypropylene notably thecrystallinity. In fact, the crystallinity of the grafted polymerdecreases with an increase in the degree of grafting. Structural changesin the polypropylene matrix due to the grafting of acrylic acid havebeen disclosed in the document Anjum, N. et al., “Surface designing ofpolypropylene by critical monitoring of the grafting conditions:structural investigations” J Appl Polym Sci, 2006.

Therefore, there is a need in the art for an improved method in order tocontrol bacterial adhesion to polyolefin substrate. Notably, there is aneed in the art for modified polyolefins whose modification route mustallow selective functionalisation of the surface. Thus the bulk matrixremains almost unaltered with its inherent physicochemicalcharacteristics.

There is also a need in the art for an easily feasible and reproduciblemethod for obtaining polyolefins having antimicrobial properties.

Thus, the invention relates to the grafting of a mixture of at least onehydrophilic monomer and one antimicrobial agent, according to a specificratio, onto a polyolefin substrate that has been exposed to ionizingradiations. The invention also relates to a method for graftingcopolymer onto a polyolefin substrate.

As used herein, “antimicrobial agents” mean polymers or monomers havingbiocide, bacteriostatic and/or repellent activity. Generally,antimicrobial agents have a high molecular weight i.e. at least 200g·mol⁻¹. Because of their size such compounds are hardly grafted. Thatmeans that only a low amount of these compounds are grafted.Furthermore, their distribution on the substrate surface area is nothomogeneous.

Surprisingly, it has been found according to the invention thatco-grafting these two specific monomers according to specificproportions, one obtains a substrate grafted with an antimicrobialcopolymer. This copolymer-grafted polyolefin substrate has a suitablehydrophilicity and a homogeneous distribution of the antimicrobialpolymer on the surface of the substrate in contact with the environment.

More surprisingly, it has been found that there is a synergic graftingeffect by co-grafting a hydrophilic monomer and an antimicrobial agent.Combining antimicrobial agents with very reactive hydrophilicunsaturated monomers increases the amount of antimicrobial agentsincorporated into the copolymer-grafted side chains. Such results arenot obtained when the grafting is carried out in two successive steps.

Furthermore, by using a specific ratio of the two specific compounds ofthe invention it is possible to modulate energetic surface properties ofthe substrate and to control bacterial adhesion. Consequently, thepresent invention enables to reduce, inhibit or delay growth ofmicro-organisms.

Additionally, no significant change due to the radiation grafting in thepolyolefin structure was observed. The grafting of the two compounds ofthe invention does not seem to lead to permeation or diffusion into thebulk matrix. The polymer chains seem to be confined on the surface.Consequently, the present invention overcomes the difficulties of theprior art by providing a copolymer-grafted polyolefin substrate withspecific properties without substantially affecting the mechanical andphysical properties of the end product material.

According to the present invention suitable hydrophilic properties areobtained notably thanks to a suitable degree of grafting. According tothe invention, a suitable degree of grafting means that the degree ofgrafting is sufficiently high to have a suitable amount of both monomersonto the surface. Additionally, the degree of grafting is sufficientlylow so that the physical properties of the substrate are not detectablymodified.

Another significant advantage of the present invention is that theantimicrobial grafted polymers are covalently bonded to the polyolefinsubstrate. Leaching is, therefore, not possible in the presentinvention. This is a significant advantage in food related applicationor medical use.

Additionally, a very good reproducibility of grafting is obtained. Thisis an advantage in order to manufacture such substrate at an industrialscale.

Therefore, it is an object of the present invention to provide a methodfor grafting a copolymer onto a polyolefin substrate. The methodcomprises the following steps:

(a) irradiating the said substrate with ionizing radiation to obtain anactivated polyolefin substrate,(b) bringing into contact the activated polyolefin substrate with amixture of at least two compounds in distilled water comprising:

-   -   (i) from 10 to 40% by volume, related to the total volume of the        reaction medium, of a hydrophilic unsaturated monomer selected        from monomers having the formula:

wherein R₁ is H or methyl, R₂ is —COON, —NH₂, —CON(R₃)₂, and R₃ is H ormethyl,

-   -   (ii) from 20 to 50% by volume, related to the total volume of        the reaction medium, of an antimicrobial agent having an average        molecular weight of at least 200 g·mol⁻¹, to thereby form a        copolymer-grafted polyolefin substrate.

The invention also relates to copolymer-grafted polyolefin substratesobtained by this method.

The invention also relates to a packaging material comprisingcopolymer-grafted polyolefin substrate according to the invention.

FIGURES

FIG. 1 shows for different ratio of comonomer mixtures [AA:METAC] thevariation of the degree of grafting with the reaction time.Preirradiation dose, 100 kGy; Energy of electron beam, 10 MeV;Temperature, 70° C.; Mohr's salt, 0.25%.

FIG. 2 shows for different ratio of comonomer mixtures [AA:METAC] thevariation of the contact angle with the degree of grafting.Preirradiation dose, 100 kGy; Energy of electron beam, 10 MeV;Temperature, 70° C.; Mohr's salt, 0.25%.

FIG. 3 shows XPS mapping of nitrogen.

FIG. 4 shows Zeta potential of ungrafted and grafted polypropylene withAA (20% and 100%), METAC (40%) and a specific ratio of comonomermixture, AA:METAC (20%:40%). Preirradiation dose, 100 kGy; Energy ofelectron beam, 10 MeV; Temperature, 70° C.; Mohr's salt, 0.25%.

FIG. 5 shows electron microscopic images of ungrafted and graftedpolypropylene showing adhesion of Listeria monocytogenes. (a) Exposedpolypropylene, (b) Grafted polypropylene with comonomer mixture,AA:METAC (20%:40%).

FIG. 6 shows the variation of the degree of grafting of acrylic acid(AA) and N,N dimethylacrylamide (DMA) with the ratio of monomer for a 30min reaction time. Preirradiation dose, 100 kGy; Energy of electronbeam, 175 KeV; Temperature, 70° C.; Mohr's salt, 0.25%.

FIG. 7 shows for different ratio of comonomer mixtures [AA:METAC] thevariation of the contact angle with the reaction time. Preirradiationdose, 100 kGy; Energy of electron beam, 175 KeV; Temperature, 70° C.;Mohr's salt, 0.25%.

FIG. 8 shows for different ratio of comonomer mixtures [DMA:METAC] thevariation of the contact angle with the reaction time. Preirradiationdose, 100 kGy; Energy of electron beam, 175 KeV; Temperature, 70° C.;Mohr's salt, 0.25%.

FIG. 9 shows a comparison of the degree of grafting carried out with 40%METAC, 20% AA, 20% DMA and a mixture of AA (20%)+METAC (40%) and amixture of DMA (20%)+METAC (40%). Preirradiation dose, 100 kGy; Energyof electron beam, 175 KeV; Temperature, 70° C.; Mohr'salt, 0.25%;Reaction Time, 40 min.

FIG. 10 shows a comparison of the water contact angle obtained with 40%METAC, 20% AA, 20% DMA and a mixture of AA (20%)+METAC (40%) and amixture of DMA (20%)+METAC (40%). Preirradiation dose, 100 kGy; Energyof electron beam, 175 KeV; Temperature, 70° C.; Mohr'salt, 0.25%;Reaction Time, 40 min.

FIG. 11 shows a comparison of the degree of grafting carried out with amixture of 20% AA, 30% AA and a mixture of AA (20%)+METAC (40%) and amixture of AA (30%)+METAC (30%). Preirradiation dose, 100 kGy; Energy ofelectron beam, 175 KeV; Temperature, 70° C.; Mohr'salt, 0.25%; ReactionTime, 30 min.

FIG. 12 shows a comparison of the degree of grafting carried out with20% DMA, 30% DMA and a mixture of DMA (20%)+METAC (40%) and a mixture ofDMA (30%)+METAC (30%). Preirradiation dose, 100 kGy; Energy of electronbeam, 175 KeV; Temperature, 70° C.; Mohr' salt, 0.25%; Reaction Time, 30min.

In the present invention, preferential polyolefin substrates areselected from the group consisting of polyethylene, polypropylene,polyisobutylene, polymethylpentene, mixtures and copolymers thereof.More preferably polyolefin is polypropylene or polyethylene. Polyolefinsubstrate can be in any shape such as a sheet, a film, a fibre, a hollowfibre or a fabric.

Hydrophilic unsaturated monomers of the present invention are preferablyacrylic acid or N,N-dimethylacrylamide.

In the context of the present invention, the antimicrobial agents arecompounds having a bacteriostatic, biocide and/or repellent activity.The mechanisms of action are different according to the researchedactivity.

Preferential monomers able to act as bacteriostatic or biocide agentsare quaternary ammonium salts (QASs). QASs are synthetic organicchemicals and are widely used in a variety of areas such asenvironmental disinfection, cosmetics, ophthalmic solution, andpharmaceutical preparation. Preferably, the QASs generally comprise apositively charged hydrophilic ammonium group and a hydrophobic alkylchain. These compounds show a biocide and/or bacteriostatic activityagainst a broad spectrum of bacteria.

Biocide QASs have an antimicrobial effect by damaging the cytoplasmicmembrane. However, such an antimicrobial effect is mainly due to thepresence of alkyl chain and their surfactant properties. The biocideactivity of QAS increases with the length of the alkyl chain carried bythe nitrogen (Ahlström and Al, 1995; 1999). Maximum activity is observedwhen the carbon number in the alkyl chain ranges from 12 to 16 (Chauhanand Al, 2004; Dizman and Al, 2004). The QASs with long alkyl chains arebetter adsorbed on cellular surface and thus damaging the cytoplasmicmembrane.

Bacteriostatic QASs have an antimicrobial effect by inhibitingreproduction of bacteria.

Biocide or bacteriostatic activity of QASs is well described in bothliterature and patents.

In order to be grafted the QASs have to be appropriatelyterminally-functionalised. That means that QASs comprise at least afunctional group capable of participating in a polymerisation reaction.The EP 0 591 024 patent describes such kind of compound. Monomers havingthe following general formula are particularly suitable.

in whichX⁻ is an anion,n is 1, 2 or 3,R₁, R₂ and R₃ are saturated C₁-C₂₀ alkyl groups, andA is an ethylene functional group capable of participating in apolymerisation reaction, preferably an acryloyloxy or methacryloyloxygroup.

Suitable example of antimicrobial monomer having a bacteriostaticactivity is [2-(Methacryloyloxy)ethyl]trimethylammonium chloride(METAC).

Antimicrobial phosphonium and sulfonium compounds can also be usedaccording to the invention.

Preferential compounds able to act as repellent agents are polyalkyleneglycols and more particularly polyethylene glycols (PEG; also referredto as polyethylene oxide, PEO).

PEGs have been investigated extensively in recent years for use asbiocompatible, protein repulsive, noninflammatory and nonimmunogenicmodifiers for drugs, proteins, enzymes, and surfaces of implantedmaterials. The basis for these extraordinary characteristics has beenattributed to the flexibility of the polymer backbone, and the volumeexclusion effect of this polymer in solution or when immobilized at asurface. Surfaces grafted with PEGs are energetically unfavourable for amicro-organism approach in aqueous solution. Consequently, the adhesionof micro-organisms is limited by steric exclusion phenomena.

The solubility of PEGs in water, as well as a number of common organicsolvents, facilitates modification by a variety of chemical reactions.Fukui and Tanaka (1976) and Fukui et al., (1987) describes the synthesisof numerous polymerizable derivatives of PEG.

The U.S. Pat. No. 5,879,709 discloses covalently crosslinkable and/orpolymerizable polyethylene glycols (PEGs). These polymerizablepolyethylene glycols are modified with a substituent which is capable ofundergoing free radical polymerization. This substituent is a moietycontaining a carbon-carbon double bond or triple bond capable of freeradical polymerization; and the substituent is linked covalently to thesaid PEG through linkages selected from ester, ether, thioether,disulfide, amide, imide, secondary amines, tertiary amines, directcarbon-carbon linkages, sulfate esters, sulfonate esters, phosphateesters, urethanes, carbonates, and the like.

Preferential polymers having a repellent activity areterminally-functionalized-polyalkylene glycol, preferably polyethyleneglycol.

Examples of such covalently polymerizable polyethylene glycols suitablefor the invention include vinyl and allyl ethers of polyethylene glycol,acrylate and methacrylate esters of polyethylene glycol, and the like.

PEGs having a wide range of molecular weights can be employed in thepractice of the present invention. According to an advantageousembodiment, the molecular weight (g·mol⁻¹) of polyalkylene glycol is inthe range of about 200 up to about 10,000 and preferably at least 2,000.Preferably the polyalkylene glycol is polyethylene glycol.

Allyl terminally functionalized polyethylene glycol compound (PEG-allyl)are particularly suitable e.g. PEG 390, PEG 2040 and PEG 10040.

The best surface properties for the polyolefin substrate are providedwith a mixture such as defined hereabove, wherein the volume ratio ofthe hydrophilic unsaturated monomer in the mixture varies from 10 to 40%by volume related to the total volume of reaction medium preferably 15to 25% by volume, and more preferably about 20% by volume and the volumeratio of the antimicrobial agent in the mixture varies from 20 to 50% byvolume related to the total volume of monomer mixture and distilledwater, preferably 35 to 45% by volume more preferably about 40%. Thereaction medium volume comprises the monomer mixture volume and thedistilled water volume.

Preferably, the concentration of hydrophilic unsaturated monomer andantimicrobial agent in the mixture is from 40 to 70%, preferably about60% by volume.

When the hydrophilic unsaturated monomer is acrylic acid, the mixturepreferably comprises:

a) from 10 to 30% by volume of acid acrylic,b) from 30 to 50% by volume of an antimicrobial agent.

When the hydrophilic unsaturated monomer is N,N-dimethylacrylamide, themixture preferably comprises:

a) from 20 to 40% by volume of N,N-dimethylacrylamide,b) from 20 to 40% by volume of an antimicrobial agent.

In order to prevent homopolymerization, the mixture further comprises ahomopolymer inhibitor selected among inorganic salts of polyvalentmetals, notably ferrous ammonium sulphate and/or copper chloride,preferably Mohr's salt.

According to an advantageous embodiment, the method further comprisesthe following steps, preferably in combination:

-   -   keeping the activated material at −80° C. prior to performing        step (b).    -   a washing step c) after grafting reaction wherein residual        monomers are removed from the surface, wherein the        copolymer-grafted polyolefin substrate is washed with distilled        water in ultrasonic water bath at 40° C. for 15 minutes,    -   a drying step d) wherein the copolymer-grafted polyolefin        substrate is dried overnight in an air oven at 40° C.

The ionizing radiations used to form the active sites on the polyolefinsubstrate should have sufficient energy to ionize the molecularstructure and to excite atomic structure. The ionizing radiation can beof any kind, but the most practical kinds comprise electrons and gammarays.

The activation of the substrate depends on different parameters such asthe dose, the energy of electron beam, the kind of substrate, thethickness or the shape of the substrate. The choice of these parameterswill be different if one wishes to activate only the surface of thesubstrate (for example a few microns) or all the volume of thesubstrate.

However, the skilled in the art is perfectly able to choose theseparameters in order to sufficiently activate the substrate to begrafted.

For example, a polypropylene substrate in the form of 1 mm thicknesssheet irradiated with a 175 KeV electron beam at a dose of 100 KGy willbe activated only on its directly exposed surface. The same sampleirradiated at the same dose but with a 10 MeV electron beam will beactivated in the whole substrate.

Furthermore, the choice of the dose can also be selected according tothe application or the substrate used. For example, a dose of 100 KGywith a 10 MeV electron beam applied on a polypropylene substrate (1 mmthickness) will damage the substrate properties whereas the same dosewill be without consequence applied with a 175 keV electron beam.Polyethylene substrate can withstand radiation doses in excess of 100kGy, without adverse effect whatever the energy of electron beam.

The polyolefin substrate is preferably irradiated during step a) withionizing radiations at a dose rate preferably in the range of about from30 to 100 kGy for a period of time sufficient for the formation of freeradical or active sites on the polyolefin surface

After the irradiation, polypropylene can be kept at low temperature toprevent deactivation of the substrate.

The monomer mixture was prepared by mixing at least the two compounds ofthe invention in distilled water. Then the mixture was added to theglass reactor along with the Mohr's salt as homopolymer inhibitor.

The step of grafting (step b) was carried out into a closed reactor. Thetemperature ranges from 20° to 80° C. and is preferably about 70° C.

The irradiated polyolefin substrate is then immersed in the mixture atselected temperature. Argon was continuously purged into the reactionmixture to create inert atmosphere. Preferably, the step b) is carriedout for a time period of at least few minutes to 2 hours and preferably15 minutes to 1 hour. After desired period, grafted polyolefin substratewas taken out and washed to avoid any traces of homopolymer.

The invention also relates to a copolymer-grafted polyolefin substratecomprising a modified surface obtained by radiation induced graftpolymerization wherein:

a) the grafted copolymer comprises:

-   -   at least a hydrophilic unsaturated monomer selected from        monomers having the formula:

wherein R₁ is H or methyl, R₂ is —COON, —NH₂, —CON(R₃)₂, and R₃ is H ormethyl,

-   -   at least an antimicrobial agent having an average molecular        weight of at least 200 g·mol⁻¹, and        b) the weight of graft per square centimetre of activated        substrate surface ranges from 0.2 to 10 mg/cm², preferably from        1 to 6 mg/cm².

Preferably, the water contact angle of the modified substrate surface isless than 40°, preferably less than 35°, more preferably less than 30°and even more preferably about 25°.

The degree of grafting was calculated in order to control the graftedamount of comonomer. The degree of grafting was calculated according tothe following equation.

$\begin{matrix}{{{Degree}\mspace{14mu} {of}\mspace{14mu} {grafting}\mspace{14mu} (\%)} = {\frac{{Wg} - {Wo}}{Wo} \times 100}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$

Where, Wo and Wg are the weights of ungrafted and grafted samples,respectively.

The degree of grafting depends on the shape of the substrate e.g. afibre, a film, a sheet. So, to avoid any ambiguity which might beintroduced by variation in substrate shape, the degree of grafting isalso expressed in term of weight of graft per square centimetre ofactivated substrate surface (Wg) using the following relationship:

$\begin{matrix}{{{Wg}\; \left( {{mg}/{cm}^{2}} \right)} = \frac{{Wg} - {Wo}}{Sa}} & \left( {{Eq}.\mspace{14mu} 2} \right)\end{matrix}$

Where, Wo and Wg are the weights of ungrafted and grafted samples, andSa is the area of the activated substrate surface respectively.

In the case of a polypropylene sheet of 1 mm thickness irradiated with a10 MeV electron beam at a dose of about 100 kGy, the energy of electronbeam is sufficient to penetrate the entire thickness of the film.Consequently, active sites are generated throughout the substrate and inparticular on the 2 faces of the sheet. Consequently, the substrate isactivated on its 2 faces (the activated area of the sheet sides areneglected).

In the case of a polypropylene sheet of 1 mm thickness irradiated with a175 keV electron beam at a dose of about 100 kGy, the energy of theelectrons is insufficient to penetrate the entire thickness of the film.Consequently, active sites are generated only on 1 face of the substrate(penetration of the electron beam is only a few microns). Consequently,the substrate is activated only on one face.

The hydrophilicity of copolymer-grafted polyolefin substrate of theinvention was investigated by water contact angle measurements.Generally, hydrophilic surface is a surface having a water contact angleless than 30°. FIGS. 2, 7, 8 and 10 clearly show that by using thespecific ratio of both compounds according to the invention, the watercontact angle of the surface is less than 35°, preferably less than 30°and more preferably about 25°. For instance, grafted polyolefinsubstrates with a mixture comprising 20% by volume of AA and 40% byvolume of METAC in distilled water have low water contact angles (about25°) and have appropriate amount of both monomers. On the contrary,grafted polyolefin substrates with a mixture comprising 50% by volume ofAA and 10% by volume of METAC have high contact angles.

Under typical manufacturing conditions the preferred values of degree ofgrafting are normally performed by using a proportion of monomer in themixture of about 60% by volume and preferably with 40% by volume ofantimicrobial agent.

Previous researches carried out on the same substrates (1 mm sheet ofpolyolefin) described in the following documents Anjum, N. and al.“Surface designing of polypropylene by critical monitoring of thegrafting conditions” J. Appl. Polym. Sci., 2006 and “Surface designingof polypropylene by critical monitoring of the grafting conditions:structural investigations” J. Appl. Polym. Sci., 2006, disclose that incase of surface modified by pure acrylic acid monomer, hydrophilicsurfaces are obtained with a very high degree of grafting. In case ofdiluted acrylic acid, water acts as the vehicle for the monomerdiffusion within the surface layers and therefore, the degree ofgrafting is high and the hydrophilicity is not convenient.

Furthermore, the grafting of antimicrobial agents onto polyolefinswithout using hydrophilic unsaturated monomers is very difficult so thedegree of grafting is very low. For example, the grafting of a mixtureof 40% by volume of METAC in distilled water leads to a degree ofgrafting of about 0.06±0.05% (FIG. 9). Additionally, the reproducibilityof the grafting is very poor.

Consequently, the grafting of a mixture of the two specific compounds ofthe invention gives very interesting results concerning the degree ofgrafting compared to results obtained by the grafting of the twocompounds separately.

The polyolefin substrates grafted with a mixture of acid acrylic and anantimicrobial agent have a weight of graft per square centimetre ofsubstrate activated surface between about 0.2 to 3 mg/cm². Thepolyolefin substrates grafted with a mixture of N,N-dimethylacrylamideand an antimicrobial agent have a weight of graft per square centimetreof substrate activated surface between about 3 to 8 mg/cm².

Zeta potential measurements have also been performed to determine thesurface charge and the isoelectric point (iep). The zeta potentials werecalculated on the basis of the streaming potential measurements and wereused to reflect the charge state of unmodified and modified polyolefinsurfaces. Isoelectric point (iep) is the point where the value of zetapotential is zero that means the total positive charges are equal to thetotal negative charges. Beyond iep, the zeta potential increases due tothe adsorption of potential determining ions, which can also reveal thesurface acidity-basicity character qualitatively.

The zeta potentials of the grafted surface can vary in a broad rangeaccording to the choice of monomers. For example, the zeta potentials atneutral and physiological water pH of a polyolefin substrate graftedwith a mixture of acid acrylic and a quaternary ammonium salt arenegative. The zeta potentials at physiological water pH of a polyolefinsubstrate grafted with a mixture of N,N-dimethylacrylamide and aquaternary ammonium salt are positive. Therefore, thanks to theinvention it is possible to modulate the surface charge of thepolyolefin substrate in function of the surface charge of themicroorganism prone to contacting the substrate.

In order to demonstrate the efficiency of the invention, adhesion testswith Listeria monocytogenes bacteria have also been carried out. In thefood industries Listeria monocytogenes is a major concern because it isresponsible for several food borne diseases. Listeria monocytogenes (LM)is a gram positive bacterium and can be fatal especially forimmunocompromised individuals and pregnant women. Apart from the rawfood, LM has also been found on various food processing surfaces.

The observation of adhesion of LM onto modified and unmodifiedpolyolefin substrate by electron microscopic images clearly shows thatthe bacterial adhesion is reduced. In fact, LM colonized the ungraftedpolyolefin substrate in a monolayer rod shape structure with highdensity. In contrast very little LM adhesion has been observed ontografted polyolefin substrate surface according to the invention.

Furthermore, the bacteria contacting polyolefin substrate grafted with amixture of 20% by volume of acid acrylic and 40% by volume of quaternaryammonium salt seem to be damaged on modified surfaces (FIG. 5). This canbe described as the modified polyolefin substrate has QAS and carboxylgroups; therefore they show antibacterial and anti-adhesion activityagainst LM.

Consequently, the copolymer-grafted polyolefin substrate of theinvention is particularly suitable for inhibiting or preventing growthof Listeria monocytogenes. Thus the invention also relates to the use ofa copolymer-grafted polyolefin substrate to inhibit or prevent growth ofListeria monocytogenes.

However, the subject of the invention is not limited to food relatedapplications. The invention can be applied in such fields aspharmaceutical products; human hygiene products; membranes;opthalmologic devices. Therefore, the invention also relates to the useof copolymer-grafted polyolefin substrate in food related applicationsand medical, pharmaceutical or cosmetic applications.

For instance, the copolymer-grafted polyolefin substrate can be apackaging material. As used herein, packaging material means anymaterial known to those of skill in the art that can be used forpackaging pharmaceutical, cosmetic, food or other consumable products.

Exemplary packaging material includes, but is not limited to,containers, vials, blister packs, bottles, tubes, inhalers, pumps, bags,tubes and any containing means.

Specific examples of product are injection solutions, eye drops,medications for internal use, medications for external use, and otherpharmaceutical products; cosmetic lotions, perfumes, cosmetic creams,and other cosmetic products; and nutritional supplement drinks,beverages, fruit juices, and other food products.

According to the invention, the packaging material and more particularlyat least the inner portion of the packaging material is made of, orcoated by a copolymer-grafted polyolefin substrate of the invention.Thus the invention also relates to a packaging material comprising acopolymer-grafted polyolefin substrate used to store or contain aconsumable product such as food, pharmaceutical, cosmetic or medicalproduct.

Experimental 1. Materials

Polypropylene (PP) of 1 mm thickness was received from GoodfellowCambridge Ltd. UK. (density, 0.9), Acrylic acid, N,N-dimethylacrylamide,METAC and Mohr's salt was supplied by Aldrich, Germany. Acrylic acid andMETAC were used without any purification. RBS-35 was supplied byChemical Products, Belgium. Distilled water was used for all theexperiments.

Before irradiation, PP samples (30×20×1 mm³) were washed with a 2%RBS-35 solution in water for 10 min at 40° C., then 5 times with tapwater at 40° C. and finally with distilled water at room temperature.Washed sample were dried overnight in an oven at 40° C.

In the examples, unless otherwise mentioned, all percentages and partsare by volume. All these values can be easily converted into weightratio thanks to the densities of the used products.

The density of used products is given for information:

AA: 1.05 g/ml, DMA: 0.962, METAC: 1.105 g/ml, PEG: 1.1-1.2 g/ml.

2. Characterisation Methods

1. Degree of Grafting

The degree of grafting is calculated according to the followingequation.

$\begin{matrix}{{{Degree}\mspace{14mu} {of}\mspace{14mu} {grafting}\mspace{14mu} (\%)} = {\frac{{Wg} - {Wo}}{Wo} \times 100}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$

where, Wo and Wg are the weights of ungrafted and grafted polypropylenesheets, respectively.The grafted weight per square centimetre of activated substrate surface(Wg) is calculated according to the following equation.

$\begin{matrix}{{{Wg}\; \left( {{mg}/{cm}^{2}} \right)} = \frac{{Wg} - {Wo}}{Sa}} & \left( {{Eq}.\mspace{14mu} 2} \right)\end{matrix}$

Where, Sa is the area of the activated substrate surface in cm².

2. Contact Angle Measurement

Contact angle measurements were carried out on KRUSS G 40 goniometer.Samples were mounted on platform and a drop of water was placed on thesurface. The contact angle was measured within 30 s of placing the dropon the sheet surface unless otherwise specified. An average of sevenmeasurements was reported.

3. Determination of the Energetic Characteristics of Modified PPSurfaces

Contact angle measurements were carried out on KRUSS (France) G 40goniometer with three pure liquids of known surface properties i.e. highpurity water (Millipore milliQ), formamide and α-bromonaphthelene(supplied by Sigma France). Samples were mounted on platform and a dropof appropriate liquid was placed on the surface. The contact angle wasmeasured within 30 s of placing the drop on the polypropylene surfaceand an average of seven measurements was reported. The surface energeticproperties of the ungrafted and grafted PP were determined by using thefollowing equation of Young-van Oss:

(1+cos θ)γL=2[(γS ^(LW) γL ^(LW))1/2+2(γS ⁺ γL ⁻)1/2+2(γS ⁻ γL⁺)1/2]  (Eq.3)

where γL is the total surface tension, γL^(LW) is the Lifshitz-van derWaals and γ⁺ and γ⁻ are the electron-acceptor/donor components of thesurface of unmodified and modified PP (S) and were estimated bymeasuring contact angles (θ) with above mentioned three pure liquids(L). The energetic characteristics of all three pure liquids arepresented in Table I. The solid surface free energy was expressed inmJ/m².

TABLE I Values of energetic characteristics components of pure liquidsPure Liquids γ^(LW) γ⁻ γ⁺ γ^(AB) γ^(T) Water 21.8 25.5 25.5 51.0 72.8α-bromonaphthalene 40.0 0.0 0.0 0.0 40.0 Formamide 39.0 39.6 2.3 19.058.0The contact angle values of α-bromonaphthalene with γ⁻ and γ⁺=0 was usedto derive γL^(LW) while water and formamide contact angles were insertedin Eq.2 to get electron donor γ⁻ and electron acceptor γ⁺ components ofthe surface free energy respectively.

4. Zeta Potential Measurements

Zeta potentials (ζ) of PP sheets were determined by streaming potentialmeasurements to get surface charge. Zeta potentials (ζ) were determinedusing a Zetacad from CAD Instrumentation®, France. The concentration ofNaCl solution was 1.5×10⁻³ M. The pH of the solution was adjusted withinthe range of 2-10 by adding KOH or HNO₃. Zeta potential was measuredthrough streaming potential method described by Van Wagenen et al.Streaming potential ΔE was measured at a driving pressure ΔP, whichvaries from 50 to 110 mBar. The measurements were repeated six times foreach pressure that means three times for one flow direction and threetimes in the reverse flow direction. The streaming potential dependsboth on the surface charge in the diffuse layer and the electrolyteproperties i.e. conductivity Ksol, viscosity η and dielectric constantD. For flat surface like PP sheets, streaming potential (ΔE) is relatedto zeta potential, so by knowing ΔE, zeta potential (ζ) can becalculated by the following equation:

$\begin{matrix}{\zeta = {\frac{4{\pi\eta}}{D}\left( {K_{{sa}\; 1} + \frac{L_{surf}}{b}} \right)\frac{\Delta \; E}{\Delta \; P}}} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$

where, Lsurf represents the specific conductance at the surface of shearand b the half distance between the two samples. The value of b was 0.05mm in our experimental setup.

5. X-ray Photoelectron Spectroscopy (XPS)

XPS measurements were made using ESCALAB 250 equipped with anonchromatized AIK∝Xray source from Electron Corporation. The analysiswas carried out under UHV of 6×10⁻⁹ mbar and for erosion 8×10⁻⁸ mbar UHVwas used. Argon flow was added in the ion gun.

6. Observation of Bacterial Adhesion by Electron Microscopy

Adhesion of Listeria monocytogenes was observed by electron microscopicimages. The strain of Listeria monocytogenes CIP 103574 (N^(o) UBHM 152)used in this study was provided by UBHM (INRA, France). Bacterial cellswere stored in a biofreezer at −80° C. prior to the experiments. Thestrains were subcultured twice, and then cultivated for 24 h at 20° C.in BHI (brain heart infusion, Oxoid) under oxygenated conditions untilthe stationary stage was reached. For the preparation of bacterialsuspension, the cells were harvested by centrifugation for 10 min at7000×g and 4° C. and then washed twice with, and resuspended in thesterile suspending liquid (1.5×10⁻³ mol·l⁻¹ NaCl).

Listeria monocytogenes cells adhering to inert surfaces were visualisedby field emission scanning electron microscopy (FESEM). Before analysis,each sample was rinsed with 150 ml of demineralised water, fixed with 3%glutaraldehyde for 1 h and washed three times with sodium cacodylate(0.2 mol·l⁻¹, pH 7.4). Further fixing with 1% osmium tetroxide for 1hour at 4° C. was the performed. The samples were washed with sodiumcacodylate and then dehydrated by passage through a graded series ofethanol/water solutions from 50 to 100%. The samples then remaineddesiccated before gold/platinum sputtering and viewing as secondaryelectron images (8 kV) with a Hitachi S4500 FESEM

EXAMPLE 1 Influence of Monomer Concentration 1. Irradiation and GraftingReaction

Electron beam (Linear electron accelerator CIRCE II) was used for theirradiation of polypropylene sheets. The energy of electron beam was 10MeV and the power was 20 kW with the speed of 0.44 m/min (IONISOSlaboratory, Orsay, France). The irradiation was carried out in air forthe dose of 100 kGy. Samples were exposed twice for the irradiation(dose of 50 kGy each). After the irradiation, polypropylene sheets werekept at −80° C. prior to the grafting reaction.

Grafting reaction was carried out in a closed reactor. The monomersolution was prepared by mixing a mixture of two monomers AA and METAC(received from Aldrich, Germany) in distilled water (monomers were usedwithout any purification). The required amount of monomer solution wasadded to the glass reactor along with the Mohr's salt as homopolymerinhibitor. The reactor was placed in an oven at 70° C. Electron beamirradiated PP sheet (100 kGy) was placed in the monomer solution in aclosed reactor. Argon was continuously purged into the reaction mixtureto create inert atmosphere. After desired period, grafted PP sample wastaken out and washed with distilled water in ultrasonic water bath at40° C. for 15 minutes to avoid any traces of homopolymer. Grafted PPsamples were dried overnight in an oven at 40° C.

Different ratio of each monomer (AA:METAC) in distilled water weretested such as 50:10, 40:20, 30:30, 20:40, 10:50 and denoted as M-1,M-2, M-3, M-4 and M-5 respectively.

For comparison, mixture comprising 40% of METAC in distilled waterdenoted METAC-40 and mixture comprising 20% of AA in distilled waterdenoted AA-20 were also tested.

2. Results

1. Degree of Grafting and Surface Hydrophilicity

The effect of each monomer composition in comonomer mixture with time onthe degree of grafting is shown in FIG. 1 and the contact anglemeasurements on virgin, exposed and grafted PP are presented in Table Iand FIG. 2.

TABLE I Values of contact angle measurements with water on virgin,exposed and grafted PP Reaction Contact Monomer time Degree of Gw AngleConcentration (%) (min) Grafting (%) (mg/cm²) (Degree) Virgin PP 0 0.0 090.0 ± 3.4 Exposed PP 0 0.0 0 89.5 ± 5.6 AA:METAC 5 1.0 0.45 59.4 ± 1.9M-1: 50:10 10 2.3 1.03 54.7 ± 0.0 15 2.5 1.12 53.3 ± 2.2 20 3.4 1.5354.6 ± 3.8 30 4.5 2.02 52.5 ± 1.7 40 4.9 2.20 50.6 ± 0.7 60 7.0 3.1550.5 ± 0.3 M-2: 40:20 5 0.8 0.36 57.6 ± 2.8 10 1.9 0.85 55.0 ± 0.8 152.7 1.21 55.0 ± 2.3 20 3.3 1.48 54.4 ± 0.0 30 4.6 2.07 54.5 ± 0.9 40 5.02.25 49.2 ± 0.1 60 6.7 3.01 47.9 ± 1.0 M-3: 30:30 5 1.1 0.49 49.2 ± 3.210 2.0 0.9 32.0 ± 0.2 30 4.2 1.89 25.6 ± 2.3 40 5.0 2.25 23.1 ± 0.4 605.9 2.65 23.3 ± 2.3 M-4: 20:40 5 0.2 0.09 60.1 ± 1.4 10 0.6 0.27 34.0 ±0.7 15 1.1 0.49 34.9 ± 4.0 20 1.6 0.72 25.1 ± 1.0 30 2.7 1.21 22.0 ± 0.740 3.6 1.62 23.9 ± 0.7 60 3.7 1.66 20.4 ± 1.1 M-5: 10:50 5 0.04 0.0270.0 ± 5.5 15 0.1 0.04 51.5 ± 4.9 20 0.3 0.13 43.4 ± 0.6 30 0.4 0.1843.0 ± 2.2 40 0.7 0.31 34.2 ± 3.9 60 1.0 0.45 26.0 ± 2.6

The degree of grafting increases with the increase in AA proportion incomonomer mixtures with time. However a decreasing trend in the degreeof grafting has been observed with the increase in METAC concentrationin comonomer mixtures. This can be clearly seen especially in thecomonomer ratio of AA:METAC (20:40) and (10:50).

The decrease in the degree of grafting may be due to the fact that whenthe proportion of METAC in comonomer mixtures increases the viscosity ofreaction medium also increases, as the viscosity of METAC is higher thanAA. It may be assumed that when the viscosity of monomer solution ishigher the monomer chains are less mobile, so less monomer is availableat the grafting sites for the grafting reaction. Hence degree ofgrafting decreases. Another reason could be that when the viscosity ofmonomer solution is higher there is no swelling of the grafted layers sothat monomer could not diffuse within the polymer bulk.

In the present investigation, the viscosity of the comonomer solution inthe presence of METAC is such that the swelling of grafted layers andmonomer diffusion through these grafted layers are inhibited, resultingto the decrease in the degree of grafting.

It could be surprising that contact angle increases as we increase theratio of AA in the comonomer mixtures. This may be due to the dilutionof monomer. When the ratio of METAC is lower the viscosity of themonomer solution is also lower, so the diffusion of monomer within thesurface layers is easier as compared to the one where the ratio of METACis higher. The increase of the viscosity slow down the diffusion ofmonomer inside the surface layers and ensure grafts on the surface ofpolypropylene. Hence decrease of the water contact angle was due to thepresence of hydrophilic METAC on the surface and no diffusion of AA.

The results in table 1 clearly demonstrate that the advantageoushydrophilicity is obtained for a ratio AA:METAC comprised between 40:20and 10:50 and more particularly 20:40. The samples grafted with thisspecific ratio show water contact angle as low as 22° and haveappropriate amount of both monomers.

2. Individual Effect of Each Monomer on the Degree of Grafting

TABLE II Values of contact angle measurements with water on virgin,exposed and grafted PP Grafted weight per unit Monomer Degree of ofactivated surface Contact Angle Concentration (%) Grafting (%) (mg/cm²)(Degree) Virgin PP 0.0 0 90.0 ± 3.4 Exposed PP 0.0 0 89.5 ± 5.6 AA-201.9 0.85 59.0 ± 6.6 METAC-40 0.09 0.04  28.0 ± 0.09 M-4 20:40 2.7 1.2122.0 ± 0.7

In the presence of comonomer mixture [AA (20%):METAC (40%)], degree ofgrafting was found to be higher as compare to the grafting, which hasbeen taken place under both monomers individually. It seems that thepresence of AA in the comonomer mixture facilitates the grafting ofMETAC.

Moreover, METAC-40 modified PP surfaces show almost similar wettabilityas M-4 modified samples. In reverse AA-20 grafted PP surfaces exhibithigher water contact angle as compare to M-4 and METAC-40 modified PPsurfaces. It may be assumed that in the grafted sample METAC is presentat the interface of the sample and AA is in the sub layers of thesurface. This may be the reason of lower value of contact angle graftedwith comonomer mixture.

3. XPS Spectrum of Virgin, Exposed and Grafted PP

TABLE III XPS elemental composition of C1s, O1s and N1s Sample Grafting(%) C1s (%) O1s (%) N1s (%) Cl2p (%) O1s/C1s N1s/C1s Virgin PP — 95.24.8 — — 0.05 — (100)*   Exposed PP — 92.7 6.4 — — 0.06 — (100)*   PPgraft with 0.03 72.8 ± 1.2 17.6 ± 0.9 5.9 ± 0.6 3.8 ± 0.7 0.23 0.08METAC (40%) (66.6)* (16.8)* (8.3)* (8.3)* (0.25)* (0.12)* PP graft with2.7 75.9 ± 1.6 18.3 ± 1.4 4.8 ± 0.2 0.3 ± .006 0.24 0.06 AA + METAC(65.6)* (20.6)* (6.8)* (6.8)* (0.31)* (0.10)* (20% + 40%) *Calculatedvalue

The spectrum of virgin PP reveals the presence of carbon signal, howeverin spite of the paraffinic nature of PP oxygen signal is also there. Thepresence of oxygen may be due to the thermo-oxidative degradation andthe surface oxidation of the polypropylene during storage or it can bedue to the environmental contamination.

Grafting of METAC leads to the origin of an additional peak of nitrogen,this shows the presence of METAC on the surface. When the grafting wasperformed in a comonomer mixture of AA (20%) and METAC (40%), the sameadditional peak is observed but the O1s content increases and N1scontent decreases. However the difference is not significant in both thespectra.

4. XPS Imaging Elemental Distribution of Nitrogen

XPS imaging enables mapping of the distribution of particular elementsor functional group on a surface. Conventional XPS provides an averagecomposition over the analyzed area. However, many sample surfaces arenot homogeneous and an element present at apparently low concentrationin the spectrum may be concentrated in one region of the area ofinterest, possibly leading to a completely different interpretation ofthe data. That is why XPS imaging on grafted PP sample to map theelemental distribution of nitrogen was carried out as shown in FIG. 3.

Each resultant image pixel represents the peak height of the imagednitrogen (N) species at that spatial position. Thus a set of images canbe quantified pixel by pixel in an analogous manner to thequantification of the spectra. This means, on the grafted sample (FIG.3), we have obtained a surface with an almost homogenous finish ofnitrogen. This indicates that in our experimental system, grafting takesplace in a homogenous manner.

5. Surface Energetic Characteristics of Modified Surface

TABLE IV Values of contact angle measurements by using three pureliquids (water, formamide and α-bromonaphthelene) on ungrafted andgrafted PP Contact Angle (degree) Sample Grafting (%) Water Formamideα-bromonaphthalene Virgin PP 0.0 90.0 75.1 45.2 Exposed PP 0.0 90.0 78.141.4 M-1 PP-g-AA:METAC 2.4 53.2 26.0 28.1 50:10 M-2 PP-g-AA:METAC 1.955.0 23.0 25.1 40:20 M-3 PP-g-AA:METAC 2.0 32.6 21.2 26.9 30:30 M-4PP-g-AA:METAC 2.3 21.9 25.2 20.7 20:40 M-5 PP-g-AA:METAC 1.2 25.5 21.530.5 10:50 PP-g-AA-20 1.8 59.0 20.0* 20.0 PP-g-METAC-40 0.07 28.0 20.0*40.0 PP-g-100% AA 8.0 28.6 20.0* 24.2 *Formamide contact angle showscomplete hydrophilicity for the PP modified with METAC and AAindividually. It is to be noted that we put 20°, as the value of contactangle to show complete hydrophilicity, as the real value was too smallor even was not possible to measure the contact angle.

TABLE V Surface energetic characteristic components of ungrafted andgrafted PP γ^(LW) γ⁺ γ⁻ Sample (mJ/m²) (mJ/m²) (mJ/m²) γ^(AB) (mJ/m²)Virgin PP 29.0 0.01 5.55 0.57 Exposed PP 30.6 0.35 7.56 3.27 PP-g-20% AA37.5 4.36 10.18 13.24 PP-g-40% METAC 31.1 3.29 43.46 23.92 PP-g-100% AA36.56 1.81 43.55 17.79 PP-g-AA:METAC 35.43 3.36 17.57 15.4 (50%:10%)PP-g-AA:METAC (40%:20%) 36.3 3.78 14.63 14.89 PP-g-AA:METAC (30%:30%)35.72 2.1 39.56 18.2 PP-g-AA:METAC (20%:40%) 37.5 1.42 47.28 16.43PP-g-AA:METAC (10%:50%) 34.82 1.94 47.5 19.21

The electron donor component γ⁻ was shown to vary greatly from 5.5 mJ/m²for unmodified to 10.18-47.28 mJ/m² for modified PP surfaces. Thissuggested that PP samples grafted with AA-20 show moderately hydrophiliccharacter, while grafting with METAC-40 exhibited hydrophilic andsurprisingly, basic character. For the grafted samples with a mixture ofthese two monomers the value of γ⁻ increases with the increase of METACratio in the comonomer mixture indicating the increase in thehydrophilicity and basic nature.

6. Potential Zeta

FIG. 4 shows Zeta potential of ungrafted and grafted polypropylene. Thevariation of zeta potential with pH indicates that H⁺ and OH⁻ ions arepotential determining ions. They are ionic species of the aqueousmedium, which actually interact with the surface. Depending on theacid-base character of the surface, the group present on the surface maygain or lose a proton according to the pH value of the aqueous phase.

TABLE VI Values of zeta potential at neutral and physiologic water pHand iep of ungrafted and grafted PP ζ (mV) at ζ (mV) at physiologicSample neutral pH water iep Virgin PP −40.0 −33.0 3.3 Exposed PP −43.0−33.0 2.6 PP-g-AA-20% −27.0 −23.0 3.7 M-4 PP-g-METAC-40 +27.5  +30.0. —PP-g-AA:METAC −16.0 −15.0 5.2 (20%:40%)

The negative zeta potential of virgin PP and exposed PP may be due tothe processing of PP at elevated temperature, which causes the thermaldegradation or partial oxidation of PP during storage and contaminationon the PP surface. In that case, the behaviour of negative charge onexposed PP would be different than the negative charge of grafted PPcaused by carboxylic groups.

The iep of comonomer mixture (M-4) grafted PP surfaces is 5.2. Atneutral and physiologic water pH, the values of zeta potentials arenegative. Here one can assume that above the iep the negatively chargedsurfaces are generated by acidic functional group such as carboxylicgroups while below the iep, presence of basic functional group isresponsible for positive charge. However, the above modified surfaceshave much stronger positive charge at acidic conditions than negativecharge at alkaline conditions. This could suggest that the modifiedsurfaces may have a higher density of METAC than carboxylic groups.These results can be correlated with the contact angle measurements,where the PP grafted with comonomer mixture (M-4) and METAC-40, exhibitalmost identical wettability, whereas PP grafted with AA-20 showsdifferent behaviour of wettability. Moreover, PP surfaces grafted withcomonomer mixture (M-4) and METAC-40 also have the identical maximumvalue of zeta potential in acidic pH.

7. Observation of LM by Electron Microscopy

Adhesion of LM was observed by electron microscopic images on unmodifiedand modified polymeric surfaces having different surfacecharacteristics.

Bacterial adhesion with solid substrate is thought to be governed as aresult of different parameters present in the suspension medium such aspositively-negatively charged ions in solution, the charge on thebacterial wall and the acid/base sites on the polymeric surface.Therefore it is important to know the surface characteristics ofListeria monocytogenes (LM) and polymeric surfaces we used for bacterialadhesion test. It can be seen that LM exhibits highly negative chargeand electron donor (γ⁻) character i.e. hydrophilic nature. According tocharacteristics of LM we have modified the PP surface by radiationgrafting of comonomer mixture.

The EM images of LM adhesion experiments performed on unmodified andmodified PP surfaces are shown in FIGS. 5 a and 5 b. The extent ofbacterial adhesion seems to be higher in the case of exposed than thePP, which is grafted with comonomer mixture (M-4). The differentadhesion trend may be explained taking into account the hydrophilicityand the presence of an acid-base interaction.

It is well known that the adhesion should be higher on hydrophobic thanon hydrophilic surfaces. We can correlate these assumptions with ourcontact angle results, which show that exposed PP has hydrophobic whilethe above grafted samples exhibit highly hydrophilic surface.

These results can also be explained that when the interacting surfacesare highly hydrophilic, the acid-base interactions lead to hydrophilicrepulsion, whereas in the case of strongly hydrophobic interactingsurface long-rang hydrophobic attraction occurs.

Comonomer grafted PP and LM have shown hydrophilic nature, leading tothe repulsive interaction, which results less adhesion. Furthermore, thecomonomer grafted PP and LM have presented higher value of electrondonor character. Therefore, we can assume the repulsive interactionbetween them, which lowers the bacterial adhesion on the grafted PP.

Additionally, FIG. 5 a shows that LM colonized the exposed PP sheetsevenly in a monolayer rod shape structure with high density. In contrastvery little LM adhesion has been observed on comonomer grafted PPsheets. Furthermore the bacterium seems to be damaged on modifiedsurfaces. This can be described as the comonomer modified PP has QAS andcarboxyl groups; therefore they show antibacterial and repulsive(anti-adhesion) activity against LM.

EXAMPLE 2 Influence of Hydrophilic Unsaturated Monomer

In this example, the water contact angles were measured immediatelyafter placing the drop on the sheet surface.

1. Irradiation and Grafting Reaction

Electron beam (Low energy electron accelerator LAB UNIT—Energy Science®)was used for the irradiation of PP sheets. The energy of electron beamwas 175 KeV, with an intensity of 5 mA. The irradiation was carried outin air for the dose of 100 kGy. After the irradiation, PP sheets werekept at −80° C. in liquid nitrogen prior to the grafting reaction.

Grafting reaction was carried out in a closed reactor. The mix monomers,water and Mohr's salt was added in the reactor placed at 70° C. in anoven during 20 minutes to heat the solution. Irradiated PP sheet wasplaced after this time, in the reactor. Argon was continuously purgedinto the reaction mixture to create inert atmosphere. After 15 minutes,grafted PP sample was taken out and placed in distilled water in sonicbath (Bioblock® Scientific 86480) at 40° C. during 10 minutes. GraftedPP sample was dried overnight in an oven at 40° C.

Mixtures comprising different ratio by volume of METAC and AA or DMAhave been tested.

2. Results

1. Degree of Grafting

FIG. 6 shows the individual effect of AA and DMA on the degree ofgrafting for a 30 min reaction time. Contrary to AA, the degree ofgrafting of DMA is proportional to the ratio of monomer in the reactionmedium. This may be due to the viscosity of DMA which is higher than theviscosity of AA and which limits its diffusion in the polyolefinsubstrate. No permeation of monomer seems occur in the polyolefinsubstrate.

FIGS. 7 and 8 shows for different ratio of comonomer mixtures [AA:METAC]and [DMA:METAC] the variation of water contact angles with the reactiontime. For a particular ratio [AA:METAC] and [DMA:METAC], the value ofwater contact angle is stabilized after a time of contact between theactivated substrate and the mixture of monomer. In the case ofpolypropylene activated according to the following conditions:Preirradiation dose, 100 kGy; Energy of electron beam, 175 KeV;Temperature, 70° C.; Mohr'salt, 0.25%; the equilibrium state is reachedfor a reaction time of about 40 minutes.

Tables VI and VII shows for different ratio of comonomer mixtures[AA:METAC] and [DMA:METAC] and for a 40 minutes reaction time thevariation of the degree of grafting and of the grafted weight per squarecentimetre of activated substrate surface. The polyolefin substratesgrafted with a mixture [AA:METAC] have weight of graft per squarecentimetre of substrate activated surface between about 0.2 to 3 mg/cm².The polyolefin substrates grafted with a mixture [DMA:METAC] have weightof graft per square centimetre of substrate activated surface betweenabout 3 to 8 mg/cm².

TABLE VII Values of degree of grafting with different ratio of comonomermixtures [AA:METAC] Monomer Degree of Grafting (%) Gw (mg/cm²) 40% AA +20% METAC 3.14 2.82 30% AA + 30% METAC 2.05 1.84 20% AA + 40% METAC 1.341.20 10% AA + 50% METAC 0.35 0.31

TABLE VIII Values of degree of grafting with different ratio ofcomonomer mixtures [AA:METAC] Monomer Degree of Grafting (%) Gw (mg/cm²)40% DMA + 20% METAC 7.79 7.01 30% DMA + 30% METAC 5.86 5.27 20% DMA +40% METAC 3.37 3.03

2. Individual Effect of Each Monomer on the Degree of Grafting

TABLE IX Values of contact angle measurements with water on grafted PPwith different ratio of comonomer mixtures [AA:METAC] and [DMA:METAC],reaction time 40 min. Degree of Gw Contact Angle Monomer Concentration(%) Grafting (%) (mg/cm²) (Degree) 20% AA 0.525 0.47 56.96 40% METAC 0 027.63 20% AA + 40% METAC 1.34 1.21 25.68 20% DMA 1.49 1.34 38.96 20%DMA + 40% METAC 3.37 3.03 25.25

FIG. 9 and table VIII shows the very low degree of grafting of METACindividually. This may be due to different parameters: the very highviscosity of METAC, the sterically hindered environment of METAC and/orthe surface properties of PP (non polar and hydrophobic substrate).

In the presence of comonomer mixture [AA (20%):METAC (40%)] or [DMA(20%):METAC (40%)], degree of grafting was found to be higher as compareto the grafting which has been taken place under both monomersindividually. It seems that the presence of very reactive hydrophiliccompounds (AA or DMA) in the comonomer mixture facilitates the graftingof METAC.

These results are also obtained in the presence of comonomer mixture [AA(30%):METAC (30%)] or [DMA (30%):METAC (30%)] (FIGS. 11 and 12).

The values of water contact angle reinforce this assumption. Indeed, thewater contact angle of PP grafted with 20% of AA or DMA in distilledwater is about 57 and 39° respectively.

The water contact angle of PP grafted with 40% of METAC in distilledwater is about 27°. The water contact angle of PP grafted with a mixtureof 40% of METAC and 20% of AA or DMA in distilled water is about 25°. Sowater contact angles of grafted PP with mixtures of [AA:METAC] and[DMA:METAC] are very close to water contact angle of PP grafted withMETAC individually. This could suggest that the METAC is preferentiallyon the surface of the substrate.

3. XPS Spectrum of Virgin, Exposed and Grafted PP

TABLE X XPS spectrum of virgin, exposed and grafted polypropylene withAA, DMA and comonomer mixtures of [AA:METAC] and [DMA:METAC] Sample C1s(%) O1s (%) O1s/C1s N1s (%) N1s/C1s Cl2p (%) Virgin PP 100*   0*  0*  —— — 95.24  4.76 0.04 PP graft with 71.4* 14.3* 0.2* 14.3* 0.2* — DMA73.69 12.47 0.17 12.73 0.7 PP graft with 67.7* 16.1*  0.23* 9.67* 0.14*6.45 DMA + METAC 72.28 13.63 0.19 9.5 0.13 4.6 (20% + 40%) PP graft with60*   40*    0.66* — — — AA 71.4  27.67 0.38 PP graft with 65.5* 20.6* 0.31* 6.8* 0.1* 6.8* AA + METAC 69.82 20.55 0.29 5.1 0.07 3.08 (20% +40%) *Calculated value

The calculated elemental composition is of the same order of magnitudethan the experimental elemental composition. This result could suggestthat there is a good correlation between the percentage of monomers inthe reaction medium and the percentage of monomers grafted onto thesubstrate surface.

4. Potential Zeta

TABLE XI Values of zeta potential and iep of ungrafted and grafted PP[AA/METAC] [DMA/METAC] Virgin PP AA DMA METAC [20/40] v/v [20/40] v/v %grafting 0 4.45 ± 0.2 2.4 ± 0.5 0.06 ± 0.05 1.4 ± 0.5 1.26 ± 0.2 Water95 ± 3   42 ± 1.6  34 ± 2.2 28 ± 8   22 ± 0.7 22.3 ± 1.5 contact angle(Hydrophilicity) ζ (mV) (at −33 −12 −16 +30 −10 1.9 pH = 6) pHi 3.3 2.22.9 >10 5.2 >10

PPs grafted with two hydrophilic monomers of the invention have verydifferent values of iep and ζ for the same hydrophilicity.

EXAMPLE 3 Influence of the Kind of Substrate 1. Irradiation and GraftingReaction

The tests were carried out with two polyolefins and one saturatedpolyester, polypropylene (PP), polyethylene (PEHD) and polyethyleneterephtalate (PET) of 1 mm thickness received from Goodfellow CambridgeLtd., UK.

Electron beam (Low energy electron accelerator LAB UNIT—Energy Science®)was used for the irradiation of PP sheets. The energy of electron beamwas 175 KeV, with an intensity of 5 mA. The irradiation was carried outin air for the dose of 100 kGy. After the irradiation, PP sheets werekept at −80° C. in liquid nitrogen prior to the grafting reaction.

Mixtures comprising 40% by volume of METAC and 20% by volume of AA areused.

Grafting reaction was carried out in a closed reactor. The mix monomers,water and Mohr's salt was added in the reactor placed at 70° C. in anoven during 20 minutes to heat the solution. Irradiated substrate wasplaced after this time, in the reactor. Argon was continuously purgedinto the reaction mixture to create inert atmosphere. After 30 minutes,grafted PP sample was taken out and placed in distilled water in sonicbath (Bioblock® Scientific 86480) at 40° C. during 10 minutes. GraftedPP sample was dried overnight in an oven at 40° C.

2. Results

TABLE XII Values of contact angle measurements with different kind ofsubstrate Grafting % Contact angle (degree) PP 1.1 ± 0.1 24.05 PE 1.7 ±0.1 26.70 PET 0.33 66.57

These results clearly show a suitable degree of grafting for thepolyolefins (polypropylene and polyethylene) in opposition to the PETfor which the degree of grafting is very low and consequently the valueof water contact angle is very high.

1. A method for grafting a copolymer onto a polyolefin substrate whereinthe said method comprises the following steps: (a) irradiating the saidsubstrate with ionizing radiation to obtain an activated polyolefinsubstrate, (b) bringing into contact the activated polyolefin substratewith a mixture of at least two compounds in distilled water comprising:(i) from 10 to 40% by volume, related to the total volume of thereaction medium, of a hydrophilic unsaturated monomer selected frommonomers having the formula:

wherein R₁ is H or methyl, R₂ is —COOH, —NH₂, —CON(R₂)₂, and R₃ is H ormethyl, (ii) from 20 to 50% by volume, related to the total volume ofthe reaction medium, of an antimicrobial agent having an averagemolecular weight of at least 200 g·mol⁻¹, to thereby form acopolymer-grafted polyolefin substrate.
 2. The method according to claim1 wherein the polyolefin substrate comprises a polyolefin selected fromthe group consisting of polyethylene, polypropylene, polyisobutylene andpolymethylpentene, mixtures and copolymers thereof.
 3. The methodaccording to claim 1 wherein the said polyolefin is polypropylene. 4.The method according to claim 1 wherein the said polyolefin ispolyethylene.
 5. The method according to claim 1 wherein the saidpolyolefin substrate is in the form of a sheet, a film, a fibre or afabric.
 6. The method according to claim 1 wherein the said hydrophilicunsaturated monomer is acrylic acid.
 7. The method according to claim 6wherein step b) is performed with a mixture of the at least twocompounds in distilled water comprising: a) from 10 to 30% by volume ofacrylic acid, b) from 30 to 50% by volume of an antimicrobial agent. 8.The method according to claim 1 wherein the said hydrophilic unsaturatedmonomer is N,N-dimethylacrylamide.
 9. The method according to claim 8wherein step b) is performed with a mixture of the at least twocompounds in distilled water comprising: a) from 20 to 40% by volume ofN,N-dimethylacrylamide, b) from 20 to 40% by volume of an antimicrobialagent.
 10. The method according to claim 1 wherein step b) is performedwith a mixture of the at least two compounds in distilled watercomprising: a) about 20% by volume of a hydrophilic unsaturated monomer,b) about 40% by volume of an antimicrobial agent.
 11. The methodaccording to claim 1 wherein the said antimicrobial agent is a compoundhaving a biocide, bacteriostatic and/or repellent activity.
 12. Themethod according to claim 11 wherein the said antimicrobial agent havinga biocide or a bacteriostatic activity is an ammonium quaternary salt.13. The method according to claim 12 wherein the said ammoniumquaternary salt is [2-(Methacryloyloxy)ethyl]trimethylammonium chloride.14. The method according to claim 11 wherein the said antimicrobialmonomer having a repellent activity isterminally-functionalized-polyalkylene glycol.
 15. The method accordingto claim 14 wherein the said polyalkylene glycol is polyethylene glycol.16. The method according to claim 14 wherein the saidterminally-functionalized-polyethylene glycol is selected from the groupof vinyl and allyl ethers of polyethylene glycol, acrylate andmethacrylate esters of polyethylene glycol.
 17. The method according toclaim 14 wherein the polyalkylene glycol has a molecular weight from 200to about 10,000.
 18. The method according to claim 17 wherein thepolyalkylene glycol is a polyethylene glycol having a molecular weightof at least 2,000.
 19. The method according to claim 1 wherein theactivated material is kept at −80° C. prior to performing step (b). 20.The method according to claim 1 which further comprises a washing stepc) after grafting reaction wherein residual monomers are removed fromthe surface, wherein the copolymer-grafted polyolefin substrate iswashed with distilled water in ultrasonic water bath at 40° C. for 15minutes.
 21. The method according to claim 1 which further comprises adrying step d) wherein the copolymer-grafted polyolefin substrate isdried overnight in an air oven at 40° C.
 22. The method according toclaim 1 wherein, at step b), the concentration of monomers in themixture ranges from 40 to 70% by volume, preferably about 60% by volume.23. The method according to claim 1 wherein, at step b), the mixturefurther comprises a homopolymer inhibitor selected among inorganic saltsof polyvalent metals, notably ferrous ammonium sulphate and/or copperchloride.
 24. The method according to claim 21 wherein the homopolymerinhibitor is Mohr's salt.
 25. The method according to claim 1 whereinstep b) is performed into a closed reactor.
 26. The method according toclaim 1 wherein step b) is performed at a temperature of about 70° C.27. The method according to claim 1 wherein step b) is performed underinert atmosphere.
 28. The method according to claim 1 wherein step (b)is carried out for a time period of at least few minutes to 2 hours andpreferably 15 minutes to 1 hour.
 29. A copolymer-grafted polyolefinsubstrate comprising a modified surface obtained by radiation inducedgraft polymerization wherein: a) the grafted copolymercomprises: atleast a hydrophilic unsaturated monomer selected from monomers havingthe formula:

wherein R₁ is H or methyl, R₂ is —COOH, —NH₂, —CON(R₃)₂, and R₃ is H ormethyl, at least an antimicrobial agent having an average molecularweight of at least 200 g·mol⁻¹, and b) the weight of graft per squarecentimetre of activated substrate surface ranges from 0.2 to 10 mg/cm²,preferably from 1 to 6 mg/cm².
 30. The polyolefin substrate according toclaim 29 wherein the water contact angle of the modified substratesurface is less than 40°, preferably less than 35°, more preferably lessthan 30° and even more preferably about 25°.
 31. The polyolefinsubstrate according to claim 29 wherein the polyolefin substratecomprises a polyolefin selected from the group consisting ofpolyethylene, polypropylene, polyisobutylene and polymethylpentene,mixtures and copolymers thereof.
 32. The polyolefin substrate accordingto claim 29 wherein the said polyolefin substrate is in the form of asheet, a film, a fibre or a fabric.
 33. The polyolefin substrateaccording to claim 29 wherein the said hydrophilic unsaturated monomeris acrylic acid.
 34. The polyolefin substrate according to claim 33wherein the weight of graft per square centimetre of activated substratesurface ranges from 0.2 to 3 mg/cm².
 35. The polyolefin substrateaccording to claim 29 wherein the said hydrophilic unsaturated monomeris N,N-dimethylacrylamide.
 36. The polyolefin substrate according toclaim 35 wherein the weight of graft per square centimetre of activatedsubstrate surface ranges from 3 to 8 mg/cm².
 37. (canceled) 38.(canceled)
 39. A packaging material comprising a copolymer-graftedpolyolefin substrate according to claim 29 used to store or contain aconsumable product such as food, pharmaceutical, cosmetic or medicalproduct.